Phosphorodiamidate-linked morpholino oligomers, or PMO, are nucleic acid analogs which bind tightly and sequence specifically to complementary RNA and are useful in modulating protein synthesis and thus gene expression. These oligomers are composed of base-pairing recognition moieties (heterocyclic bases) supported by a morpholino backbone system. Morpholino subunits for use in synthesizing such oligomers can be prepared easily from the corresponding ribonucleosides, which are readily available and inexpensive precursors (see e.g. Summerton and Weller, 1993, 1997).
During such synthesis, as in conventional oligonucleotide synthesis, the functional groups on the heterocyclic bases are typically masked to prevent interference in the synthetic transformations. For example, activation of the N-tritylated morpholino monomer (1a-f; FIG. 1) entails reaction of the 5′-hydroxyl with a suitable phosphoramido dichloridate to form the activated subunit 2a-f. At large scale (50-100 Gallon reactor), the crude activated subunit is generally contaminated with a high level of by-products. Following chromatographic purification, the activated subunit is isolated in about 50% yield for A, C, I, T, U and their protected forms, but only in about 5% yield for the activated singly protected G subunit, which is believed to be due to the presence of the unprotected O6 oxygen.
The O6-unprotected guanine subunit also gives rise to side reactions at the oligomer stage. For example, the O6 oxygen can react with activated subunit during coupling steps, to form O6-phosphorylated or derivative species, and during final cleavage of the base protecting groups with ammonia, ammonia can react at C6 to displace these species, giving a diaminopurine derivative. Such impurities are difficult to remove by chromatography, and cause a large loss in yield.
Various protection schemes have been proposed in the art to reduce side reactions of unprotected guanine O6 positions in conventional oligonucleotide synthesis (see e.g. Gough et al. 1979; Reese et al. 1981, 1984; Jones et al. 1982A, 1982B). However, these protocols were largely unsuccessful when applied to PMO synthesis. Accordingly, improved methods were sought to increase yield and purity in PMO synthesis, particularly in the use of G morpholino subunits.